Increased Prostaglandin E2 Release Research Articles

Increased Prostaglandin E2Release and Activated Akt/β-Catenin Signaling Pathway Occur after Opioid Withdrawal in Rat Spinal Cord

Prostaglandin E(2) is an important spinal modulator of nociception. However, the effects of chronic opioid administration and withdrawal on prostaglandin E(2) release and associated signaling pathways in the spinal cord are generally unknown. This study sought to examine these effects using a spinal microdialysis technique in a model of chronic morphine administration and withdrawal in the rat. The authors found that spinal prostaglandin E(2) release was unaffected by chronic morphine treatment but was significantly increased during withdrawal. Recurrent withdrawal did not further enhance this release. The authors also found up-regulation of cyclooxygenase-2 expression and phosphorylation of protein kinase Akt at Ser-473 in response to opioid withdrawal. In addition, they demonstrated that beta-catenin, a transcription factor downstream of Akt, was induced during morphine withdrawal, particularly during recurrent withdrawal. These results suggest that opioid withdrawal activates signaling pathways associated with neuronal survival and transcriptional control, two processes implicated in neuronal development and synaptic plasticity.

Metallic but not ceramic wear particles increase prostaglandin E2 release and interleukin-1β gene expression in human blood monocytes in vitro

In this study the potential of clinically relevant alumina ceramic and metal wear particles to induce an in vitro inflammatory response was assessed in human monocytes and lymphocytes isolated from healthy donors by measuring pro-staglandin E2 (PGE2) levels and mRNA expression of various pro-inflammatory cytokines. Bacterial lipopolysaccharide (LPS) was used as positive control. LPS significantly increased PGE2 levels in the incubation medium of monocyte cultures after 24 h. Alumina had no effect on PGE2 production, whereas metals induced a concentration-dependent increase in PGE2 release, that was statistically significant at the dose of 0.1 mg/ ml. In lymphocytes, LPS elicited a weak but significant increase in PGE2 release, whereas both alumina and metals did not modify PGE2 amounts at any of the concentrations tested. The gene expression of a number of pro- and anti-inflammatory cytokines was assessed in monocytes and lymphocytes exposed to LPS, 0.1 mg/ml alumina or 0.1 mg/ml metals for 24 h. In monocytes, LPS caused a 2-fold increase in interleukin-1β (IL-1β) mRNA levels. The exposure of monocytes to metals resulted in a selective increase in IL-1β mRNA accumulation (+48% compared to control). By contrast, alumina did not modify IL-1β mRNA levels. None of the test substances elicited any response on purified lymphocyte population. These findings suggest that PGE2 production and IL-1 mRNA expression are a reliable marker to study the pro-inflammatory effects of wear debris in vitro. The lower activity of alumina compared to metals suggests that the former should be pre-ferred in implants for its favorable biological and mechanical behavior.

Activation of sPLA2‐IIA and PGE2 production by high mobility group protein B1 in vascular smooth muscle cells sensitized by IL‐1β

Lipid mediators such as prostaglandin E2 (PGE2) play a central role during atherogenesis as a consequence of inflammation. PGE2 is produced from phospholipids by a cascade of enzymatic reactions involving phospholipase A2 (PLA2), cyclooxygenase (COX), and prostaglandin E synthase (PGES). It is released by several cell types, including vascular smooth muscle cells (VSMCs). Recent work has shown that the secretory PLA2-IIA (sPLA2-IIA), the most abundant isoform of secreted PLA2 in VSMCs, acts as a potent cytokine and activates VSMCs through a positive feedback loop. High mobility group protein 1 (HMGB1), also known as amphoterin, is a ubiquitous protein that plays various roles in the nucleus. HMGB1 is released by necrotic cells and by immune cells in response to various inflammatory mediators and acts as a potent proinflammatory cytokine. The present study investigates the role of HMGB1 in the activation of sPLA2-IIA expression and PGE2 production in VSMCs. Recombinant HMGB1 slightly activated the sPLA2-IIA, COX-2, and mPGES-1 genes but dramatically stimulated these genes in VSMCs that had been incubated with the proinflammatory cytokine IL-1beta for 24 h. This effect was accompanied by significantly increased PGE2 release. Induction of the three known receptors of HMGB1, namely RAGE, TLR-2, and TLR-4, by IL-1beta suggests that proinflammatory cytokines sensitize VSMCs to HMGB1. This provides new insights into the role of HMGB1 in VSMCs, suggesting it may be essential for the progression of atherosclerosis.

Prostaglandin E2 production and viability of cells cultured in contact with freshly mixed endodontic materials

To determine whether commonly used endodontic sealers could either induce or increase the release of prostaglandin E2 (PGE2) when in contact with cell types found in the periapical tissues. Freshly mixed samples of Roth 801 sealer, Sealapex and ProRoot mineral trioxide aggregate (MTA) were placed in contact with cultured macrophages and fibroblasts for 24 h. The supernatant from the cultures was assayed for PGE2 using enzyme-linked immunosorbent assay. Cell viability counts were made. As a positive control, similar cultures were also exposed to lipopolysaccharide and the supernatant analysed for PGE2. Data were compared by anova. The three materials examined in these experiments did not stimulate increased PGE2 release from either of the cell lines. In control cultures, lipopolysaccharide increased PGE2 release from macrophages but not from fibroblasts. Viability counts revealed that, whilst Roth 801 sealer caused some cell death in both fibroblasts and macrophages, Sealapex led to cell death only in the macrophage cultures. ProRoot MTA did not lead to statistically significant cell death in either culture. Under 24-h culture conditions, the three freshly mixed test materials did not increase directly either production or release of PGE2 from either macrophages or gingival fibroblasts. Roth 801 decreased cell viability counts for both fibroblasts and macrophages. Sealapex decreases macrophage viability. ProRoot MTA did not affect viability in either cell line.

IL-17 suppresses TNF-α–induced CCL27 production through induction of COX-2 in human keratinocytes

The chemokine CCL27 attracts skin-homing T cells. CCL27 production by keratinocytes is dependent on nuclear factor kappaB (NF-kappaB) activity and enhanced in lesions of patients with atopic dermatitis, psoriasis vulgaris, or allergic contact dermatitis. IL-17 is released from activated memory T cells and modulates skin inflammation. We examined the in vitro effects of IL-17 on TNF-alpha-induced CCL27 production in human keratinocytes. Keratinocytes were incubated with TNF-alpha, IL-17, or both. CCL27 secretion and mRNA levels were analyzed by means of ELISA and RT-PCR, respectively. COX-2 promoter and NF-kappaB activities were analyzed by using luciferase assays. COX-2 protein levels were analyzed by means of Western blotting. IL-17 suppressed TNF-alpha-induced CCL27 secretion and mRNA expression and NF-kappaB activity in keratinocytes. The COX-2 inhibitor NS398 counteracted the effects of IL-17, and prostaglandin E(2) prevented counteraction by NS398. IL-17 alone or synergistically with TNF-alpha increased prostaglandin E(2) release from keratinocytes, and the increase was suppressed by NS398. IL-17 alone or synergistically with TNF-alpha increased COX-2 mRNA and protein levels, promoter activity, and mRNA stability. The stimulatory effects of IL-17 on COX-2 expression were suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) kinase. IL-17 alone or synergistically with TNF-alpha induced dual phosphorylation of p38 MAPK and ERK. IL-17 might suppress TNF-alpha-induced CCL27 production by inhibiting NF-kappaB through induction of COX-2. The induction of COX-2 might be mediated by activation of p38 MAPK and ERK. T cell-derived IL-17 might alleviate T-cell skin infiltration through inhibition of CCL27 production.

Isoform-specific effects of apolipoprotein E on secretion of inflammatory mediators in adult rat microglia

Inflammation mediated by activated microglia cells has been shown to contribute to the pathogenesis of Alzheimer disease (AD) [1]. Microglia are the immune cells in the central nervous system, and when activated they secrete the lipid-derived mediator prostaglandin E2 (PGE2), the cytokine interleukin-1beta (IL-1beta), and other inflammatory mediators. Apolipoprotein E isoform 4 (apoE4), coded for by the gene APOE4 (epsilon4), has been shown to correlate with higher risk of onset of AD, as well as with increased severity of other diseases with a neuroinflammatory component. This study investigated isoform-specific effects of apoE on the regulation of PGE2, COX2, and IL-1beta expression. Two physiologically relevant preparations of apoE displayed an isoform-specific effect on inflammation in primary adult microglia cultured from adult rat brain cortex. Specifically, apoE4 alone, but not the more common isoform apoE3, stimulated secretion of PGE2 and IL-1beta. The increase in PGE2 release stimulated by apoE4 was not accompanied by the upregulation of the COX-2 enzyme in microglia.

Differential effects of IL-10 on prostaglandin H synthase-2 expression and prostaglandin E2 biosynthesis between spleen and bone marrow macrophages

Different populations of mononuclear phagocytes (MO) show considerable diversity of cellular function including prostaglandin E2 (PGE2) biosynthesis. Certain bacterial components enhance PGE2 biosynthesis differentially in selected populations of MO. Interleukin (IL)-10 is proposed to inhibit modulation of PGE2 biosynthesis by down-regulating prostaglandin G/H synthase-2 (PGHS-2) expression. To assess whether IL-10 regulates PGE2 biosynthesis and PGHS-2 expression, splenic and bone marrow MO were isolated from IL-10-deficient (IL-10(-/-)), C57Bl/6 [wild-type (WT) control], and Balb/c (comparison control) mice and were treated with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma) as a model of bacterial inflammation. LPS-induced PGHS-2 expression was similar for splenic MO isolated from the three strains of mice. However, PGE2 released by LPS-treated splenic MO was significantly higher in IL-10(-/-) and Balb/c than in WT cells. In the presence of LPS and IFN-gamma, PGHS-2 expression and PGE2 release by IL-10(-/-) and Balb/c splenic MO were enhanced compared with stimulation with LPS alone or IFN-gamma alone. However, there was no significant increase in PGE2 release from WT splenic MO treated with LPS plus IFN-gamma despite increased PGHS-2 expression. In sharp contrast, PGHS-2 expression and PGE2 release by bone marrow MO were greatly enhanced in IL-10(-/-) cells compared with control cells. Our results indicate that IL-10 regulation of MO PGE2 biosynthesis and PGHS-2 expression is compartment-dependent and that PGE2 production is not linked directly to PGHS-2 levels. Furthermore, our findings emphasize strain-specific differences between C57Bl/6 and Balb/c mice, and Balb/c appears more similar to the IL-10(-/-) than to the C57Bl/6 with respect to prostanoid production.

Platelet‐derived growth factor‐induced arachidonic acid release for enhancement of prostaglandin E2 synthesis in human gingival fibroblasts pretreated with interleukin‐1β

Platelet-derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E(2) (PGE(2)) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time- and dose-dependent manner, and simultaneously induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), but less provoked PGE(2) release and cyclooxygenase-2 (COX-2) mRNA expression. When [Ca(2+)](i) was increased by Ca(2+)-mobilizing reagents, arachidonic acid release was increased. The PDGF-induced arachidonic acid release and increase in [Ca(2+)](i) were prevented by a tyrosine kinase inhibitor. On the other hand, in the HGF pre-stimulated with interleukin-1beta (IL-1beta), PDGF clearly increased PGE(2) release. The PDGF-induced PGE(2) release was inhibited by a tyrosine kinase inhibitor. In the HGF pretreated with IL-1beta, arachidonic acid strongly enhanced PGE(2) release and COX-2 mRNA expression. These results suggest that PDGF stimulates arachidonic acid release by the increase in [Ca(2+)](i) via tyrosine kinase activation, and which contributes to PGE(2) production via COX-2 expression in HGF primed with IL-1beta.

1716: Stretch-Induced Increase in Prostaglandin E2 Release from Bladder Urothelium in Spinal Rats

You have accessJournal of UrologyDiscussed Poster,Wednesday, May 12, 2004,8:00 am - 12:00 pm1 Apr 20041716: Stretch-Induced Increase in Prostaglandin E2 Release from Bladder Urothelium in Spinal Rats Masaki Yoshida, Akita Inadome, Yutaka Miyamoto, Koichi Masunaga, Hitoshi Iwashita, Masayuki Otani, Koichi Miyamae, Yutaka Sugiyama, and Shoichi Ueda Masaki YoshidaMasaki Yoshida More articles by this author , Akita InadomeAkita Inadome More articles by this author , Yutaka MiyamotoYutaka Miyamoto More articles by this author , Koichi MasunagaKoichi Masunaga More articles by this author , Hitoshi IwashitaHitoshi Iwashita More articles by this author , Masayuki OtaniMasayuki Otani More articles by this author , Koichi MiyamaeKoichi Miyamae More articles by this author , Yutaka SugiyamaYutaka Sugiyama More articles by this author , and Shoichi UedaShoichi Ueda More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)38908-0AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "1716: Stretch-Induced Increase in Prostaglandin E2 Release from Bladder Urothelium in Spinal Rats." The Journal of Urology, 171(4S), p. 454 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 171Issue 4SApril 2004Page: 454 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Masaki Yoshida More articles by this author Akita Inadome More articles by this author Yutaka Miyamoto More articles by this author Koichi Masunaga More articles by this author Hitoshi Iwashita More articles by this author Masayuki Otani More articles by this author Koichi Miyamae More articles by this author Yutaka Sugiyama More articles by this author Shoichi Ueda More articles by this author Expand All Advertisement PDF DownloadLoading ...

Effect of Nitric Oxide on Arachidonic Acid Release from Human Amnion-like WISH Cells

In order to clarify the possible interactions between nitric oxide (NO) and arachidonic acid (AA) pathways, human amnion-like WISH cells were perifused to measure the effects of the following substances on [3H]arachidonic acid release: (1) sodium nitroprusside (SNP), a nitric oxide donor; (2) 1,1,1-trifluoromethyl-6,9,12,15-heicosatetraen-2-one, a cytosolic phospholipase A2 (cPLA2) inhibitor; (3)l -arginine, the substrate of nitric oxide synthase (NOS); (4) 3-(5′-Hydroxymethyl-2′-furyl)-1-benzylindazole and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one, activator and inhibitor of soluble guanylyl cyclase, respectively; (5) a membrane-permeable non-hydrolyzable analogue of guanosine-3′,5′-cyclic monophosphate (cGMP). Furthermore, the effect of SNP on prostaglandin E2 (PGE2) release was tested. Exogenous and endogenous NO, as well as the guanylyl cyclase activator and cGMP analogue, significantly increased [3H]arachidonic acid release. Both soluble guanylyl cyclase and PLA2 inhibitors counteracted SNP response. Exogenous NO increased PGE2 release, although to a much lesser degree compared with arachidonic acid release.Our results indicate that NO stimulates AA release in WISH cells by activating PLA2 through a cyclic GMP-dependent mechanism.

Effect of Astragali radix extract on lipopolysaccharide-induced inflammation in human amnion.

The effects of Astragali radix extract on interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha productions, prostaglandin E2 (PGE2) biosynthesis, and leukotriene C4 (LTC4) production from lipopolysaccharide (LPS)-stimulated human amnion cells were investigated. Amnion cells produced detectable amounts of both IL-6 and TNF-alpha under LPS-stimulated conditions. Astragalus extract inhibited the production of IL-6. However, TNF-alpha production was not inhibited by the extract on L929 cytotoxicity assay. Treatment of amnion cells with LPS for up to 24 h resulted in an increase in PGE2 release in a concentration- and time-dependent manner. The extract (150 mg/ml) significantly inhibited the output of PGE2 by amnion cells (p<0.01). The arachidonate lipoxygenase metabolite (LTC4) was increased by LPS treatment of amnion cells. Astragalus extract (30 mg/ml) inhibited LTC4 production by approximately 65% throughout the culture period. These results suggest that Astragali radix extract may have a role in inhibiting bacterial infection-associated preterm labor by suppressing the productions of IL-6, PGE2, and LTC4 by human amnion cells.

Glucocorticoids and TGF-beta1 synergize in augmenting fibroblast mediated contraction of collagen gels.

TGF-beta plays a central role in the initiation and progression of pulmonary fibrosis. Glucocorticoids are frequently used to treat fibrotic diseases, but beneficial effects are often modest. Both TGF-beta and glucocorticoids have been reported to increase fibroblast contraction of native collagen gels, a model of fibrotic tissue remodeling. Therefore, we sought to determine how glucocorticoids interact with TGF-beta in this system. In this study, human fetal lung fibroblasts (HFL-1) were pretreated with or without TGF-beta for 72 h before they were cast into type I collagen gels. Various concentrations of glucocorticoids (budesonide or hydrocortisone) were added at the time of casting. Gel size was then monitored at different times after gel release. The surrounding media were collected for the assay of prostaglandin E2 (PGE2) and the cell lysates were analyzed for cyclooxygenase (COX) expression by immunoblot. Glucocorticoids alone significantly enhanced fibroblast-mediated contraction of collagen gels (P < 0.01) and dose-dependently inhibited PGE2 release by HFL-1 fibroblasts. TGF-beta significantly augmented gel contraction but also induced a 30% increase in PGE2 release and increased the expression of COX-1. Glucocorticoids inhibited TGF-beta1 induced-PGE2 release, and enhanced TGF-beta augmented gel contraction without significantly affecting TGF-beta augmented COX-1 expression. Indomethacin, a COX inhibitor, increased TGF-beta augmented gel contraction but had no further effect when added together with glucocorticoids. Thus, glucocorticoids can synergize with TGF-beta in augmenting fibroblast mediated collagen gel contraction through the inhibition of PGE2 production. Such interactions between glucocorticoids and TGF-beta may account, in part, for the lack of response of fibrotic diseases to glucocorticoids.

Cytotoxicity of Root Canal Filling Materials to Three Different Human Cell Lines

The aim of this study was to investigate the biological compatibility of five root canal sealers (Sealapex, Endion, Super-EBA, Ketac-Endo, and AH Plus) and regular and calcium hydroxide-based gutta-percha in three different human cell lines. Cultures without root canal sealers were used as controls. Cell growth, cell morphology, cell viability, protein content of the cells, and prostaglandin E2 (PGE2) release were used as parameters to determine the cytotoxicity of the materials. The protein content of the three cell lines--nasal fibroblasts, gingival fibroblasts, and epithelial tumor cells--was significantly reduced (p < or = 0.001) by all materials tested. Determinations of PGE2 release showed significant material specific differences. No significant increased PGE2 release values were found with Sealapex, Endion, and Super-EBA. On the contrary significantly increased PGE2 levels were measured with Ketac Endo, AH Plus, regular and calcium hydroxide-based gutta-percha points (p < or = 0.001).

Signaling pathways involved in lipopolysaccharide stimulation of prostaglandin production by rat hypothalamic astroglial cells

The aim of this study was to investigate the mechanisms through which bacterial lipopolysaccharide (LPS) stimulates prostaglandin (PG) production in rat hypothalamic astroglial cells in vitro. The latter were treated with LPS alone or LPS plus antagonists of the interleukin-1 (IL-1) and nitric oxide (NO) pathways, and the subsequent changes in cyclooxygenase (COX) activity were monitored by measuring a COX end-product, prostaglandin E2 (PGE2), released into the incubation medium. LPS produced a concentration-dependent increase in PGE2 release from astroglia after 24 h incubation; experiments with selective antagonists showed that the increase in PGE2 release induced by LPS may be, at least in part, mediated by IL-1 and NO.

The effect of NSAIDs and a COX-2 specific inhibitor on Helicobacter pylori-induced PGE2 and HGF in human gastric fibroblasts.

There is compelling evidence for the pivotal role of Helicobacter pylori in the pathogenesis of gastrointestinal ulcer disease. However, despite the bacterium's toxicity, the majority of H. pylori infections are not accompanied by gastric ulcers. This implies the existence of a host mechanism offsetting H. pylori toxicity. To evaluate gastric fibroblasts' expression of hepatocyte growth factor (HGF), which is known to facilitate gastric ulcer healing, in the presence of H. pylori; to compare the effect on H. pylori-induced HGF expression of a COX-2 selective inhibitor with that of nonselective nonsteroidal anti-inflammatory drugs Human gastric fibroblasts were cultured from human gastric mucosa obtained at surgery. Prostaglandin E2 (PGE2) and HGF were measured by EIA. The expression of COX-2 mRNA was assessed by the TaqMan quantitative RT-PCR system. H. pylori increased PGE2 release in gastric fibroblasts. H. pylori induced expression of COX-2 mRNA, which indicates that PG induction by H. pylori is through COX-2. Sulindac sulphide, etodolac and NS 398 all inhibited H. pylori-induced PGE2 release to the same extent. These agents also inhibited H. pylori-induced HGF release. Gastric fibroblasts produce PG and HGF in response to the presence of H. pylori, which may be considered part of the human body's defensive reaction to H. pylori toxicity. This defensive mechanism is inhibited not only by COX-2 nonselective NSAIDs but also by a COX-2 selective inhibitor. These findings indicate the importance of COX-2 in chronic H. pylori infection.

Effect of Surface Roughness and Composition on Costochondral Chondrocytes is Dependent on Cell Maturation State

During endochondral bone formatin, as occurs in fracture healing, chondrocytes are one of the first cells to see an implant surface. We tested the hypothesis that chemical composition and surface roughness affect chondrocyte differentiation matrix synthesis, and local factor production and that the nature of the response is dependent on the state of maturation of the cells. To do this, we harvested rat growth zone and resting zone chondrocytes and examined their tesponce to smooth and rough disk surfaces manufactured from either commercially pure titanium or titanium alloy. profilometry, scanning electron microscopy, Auger spectroscopy, and Fourier transform infrared spectroscopy were used to characterize the surfaces. Average roughness values were 0.22 μm for smooth titanium surfaces, 0.23 μm for smooth titanium alloy surfaces, 4.24 μm for rough titanium surfaces. and 3.20 μm for rough titanium alloy surface. Cells were grown on the different disk surfaces until the cultures had reached confluence on plastic. The effect of the surfaces was determined by assaying cell number and [3H]thymidine incorporation as measurs of cell proliferatin, cell layer and cell alkaline phosphatase specific activity as markers of differentiation, and collagen production and [35S]sulfate incorporation as indicators of extracellular matrix production. In addition, the synthesis of prostaglandin E2 and transforming growth factor-β were examined to measure changes in local factor synthesis. In growth Zone and resting Zone cultures, cell number and [3H]thymidine incorporation were decreased on rough surfaces: however, this effect was greater on commercially pure titanium surfaces. Cell layer and cell alkaline phosphatase specific activity were decreased in resting zone cells grown on rough surfaces. Cell alkaline phosphatase specific activity in growth zone cells was decreased on rough surfaces, whereas cell layer alkaline phosphatase specific activity was increased only in growth zone cells growth on rough commercially pure titanium surfaces, Resting Zone cell collagen production was decreased only on rough commercially pure titanium, whereas in growth zone cells, collagen production was increased. Increased prostaglandin E2 release into the media was found for growth zone and resting zone cell cultures on the disks with rough surfaces. The observed effect was greater on rough commercially pure titanium. Production of transforming growth factor-β by resting zones was similarly affected. whereas an increase in its production by growth zone cells was measured only on rough commercially pure titanium. These results indicate that surface roughness affects chondrocyte proliferation, differentiation, matrix synthesis. and local factor production and that these parameters are also affected by chemical composition. Furthermore. the nature and extent of the cell response is dependent on cell maturation. The overriding variable in response to an implant material, however, appears to be roughness of the surface.

Induction of cyclooxygenase-2 expression in human myometrial smooth muscle cells by interleukin-1beta: involvement of p38 mitogen-activated protein kinase.

1. Human myometrial smooth muscle cells (HMSMCs) in culture were exposed to recombinant human interleukin-1beta (IL-1beta, 10 ng ml-1) for 1 to 24 h. Cyclooxygenase-2 (COX-2) mRNA and protein were rapidly induced, with expression sustained at 24 h. 2. Cycloheximide (10 microg ml-1, 6 h) blocked IL-1beta-induced COX-2 protein expression and super-induced COX-2 mRNA expression. Induction of COX-2 mRNA and protein was blocked by dexamethasone (1 microm, 6 h). 3. IL-1beta-induced COX-2 expression was accompanied by a 3-fold increase of prostaglandin E2 release into the culture medium. 4. IL-1beta induced a transient (5-30 min) activation of p42/44 and p38 mitogen-activated protein kinase (MAPK) enzymes in HMSMCs. Activity of p38 MAPK was monitored by in-gel activity of its substrate MAP kinase-activated protein kinase-2 (MAPKAP kinase-2). Induction of MAPKAP kinase-2 activity was prevented by the p38 MAPK inhibitor SB 203580 (10 microm, 5-30 min). 5. COX-2 protein expression detected after 6 h IL-1beta stimulation was blocked by SB 203580 (10 microM). Exposure of HMSMCs to 10 ng ml-1 IL-1beta for only 30 min induced a level of COX-2 protein expression at 6 h culture similar to that detected in cells exposed to the cytokine for 6 h. 6. Exposure of cells to SB 203580 (10 microM during only the first 30 min of IL-1beta stimulation was effective in blocking COX-2 protein expression assayed after 6 h in culture. 7. This study has established that a transient activation of the p38 MAPK cascade is involved in IL-1beta-stimulated COX-2 expression in human myometrial smooth muscle cells. Induction of COX-2 by IL-1beta in HMSMCs provides support for the hypothesis that autocrine prostaglandin signalling in the myometrium, initiated by elevated intrauterine cytokine concentrations, plays a role in regulating myometrial contractility during labour.

The production of interleukin-1beta from human fetal membranes is not obligatory for increased prostaglandin output.

Bacterial endotoxin increased the expression of mRNA (maximal after 4 hr) for interleukin-1beta (IL-1beta) and the release of mature protein from intact human fetal membranes. In contrast, the change in expression of mRNA for type 2 cyclo-oxygenase (COX-2) was biphasic, with peaks after 0.5-1 hr and after 8 hr of culture. An antibody to IL-1beta was without effect after 4 hr of culture, inhibited endotoxin-stimulated prostaglandin E2 (PGE2) production after 8 hr of culture, and caused a parallel decrease in the expression of mRNA for COX-2. We conclude that endotoxin induced the expression of COX-2 through IL-1beta-independent and IL-1beta-dependent mechanisms, and these differences are time dependent. Corticotrophin-releasing hormone (CRH) or platelet-activating factor (PAF) also increased the expression of mRNA for IL-1beta and the release of IL-1beta from some, but not all, fetal membranes. The antibody to IL-1beta did not affect CRH-stimulated or PAF-stimulated PGE2 production or COX-2 expression. We conclude that CRH and PAF can induce the expression of IL-1beta, but this is not obligatory for increased PGE2 release, and the effect of these stimuli on COX-2 expression is a direct, IL-1beta-independent effect.

Effect of surface roughness and composition on costochondral chondrocytes is dependent on cell maturation state.

During endochondral bone formation, as occurs in fracture healing, chondrocytes are one of the first cells to see an implant surface. We tested the hypothesis that chemical composition and surface roughness affect chondrocyte differentiation, matrix synthesis, and local factor production and that the nature of the response is dependent on the state of maturation of the cells. To do this, we harvested rat growth zone and resting zone chondrocytes and examined their response to smooth and rough disk surfaces manufactured from either commercially pure titanium or titanium alloy. Profilometry, scanning electron microscopy, Auger spectroscopy, and Fourier transform infrared spectroscopy were used to characterize the surfaces. Average roughness values were 0.22 microm for smooth titanium surfaces, 0.23 microm for smooth titanium alloy surfaces, 4.24 microm for rough titanium surfaces, and 3.20 microm for rough titanium alloy surfaces. Cells were grown on the different disk surfaces until the cultures had reached confluence on plastic. The effect of the surfaces was determined by assaying cell number and [3H]thymidine incorporation as measures of cell proliferation, cell layer and cell alkaline phosphatase specific activity as markers of differentiation, and collagen production and [35S]sulfate incorporation as indicators of extracellular matrix production. In addition, the synthesis of prostaglandin E2 and transforming growth factor-beta were examined to measure changes in local factor synthesis. In growth zone and resting zone cultures, cell number and [3H]thymidine incorporation were decreased on rough surfaces; however, this effect was greater on commercially pure titanium surfaces. Cell layer and cell alkaline phosphatase specific activity were decreased in resting zone cells grown on rough surfaces. Cell alkaline phosphatase specific activity in growth zone cells was decreased on rough surfaces, whereas cell layer alkaline phosphatase specific activity was increased only in growth zone cells grown on rough commercially pure titanium surfaces. Resting zone cell collagen production was decreased only on rough commercially pure titanium, whereas in growth zone cells, collagen production was increased. Increased prostaglandin E2 release into the media was found for growth zone and resting zone cell cultures on the disks with rough surfaces. The observed effect was greater on rough commercially pure titanium. Production of transforming growth factor-beta by resting zones was similarly affected, whereas an increase in its production by growth zone cells was measured only on rough commercially pure titanium. These results indicate that surface roughness affects chondrocyte proliferation, differentiation, matrix synthesis, and local factor production and that these parameters are also affected by chemical composition. Furthermore, the nature and extent of the cell response is dependent on cell maturation. The overriding variable in response to an implant material, however, appears to be roughness of the surface.

Induction of cyclo-oxygenase-2 expression by methyl arachidonyl fluorophosphonate in murine J774 macrophages: roles of protein kinase C, ERKs and p38 MAPK.

1. Methyl arachidonyl fluorophosphonate (MAFP), an inhibitor of phospholipase A2 (PLA2), has been widely used to assess the roles of PLA2 in various cell functions. Here, we report on a novel action of this compound at concentrations similar to those used for PLA2 inhibition. 2. The murine macrophage J774 released a large amount of prostaglandin E2 (PGE2) by MAFP (1-30 microM), which was abolished by indomethacin and NS-398 but not by valeryl salicylate, and results from increased cyclo-oxygenase-2 (COX-2) protein levels and gene expression. 3. This PGE2 release was blocked by inhibitors of tyrosine kinase (genistein), protein kinase C (PKC) (Ro 31-8220, Go 6976 or LY 379196), mitogen-activated protein kinase kinase (MEK) (PD 098059) or p38 mitogen-activated protein kinase (MAPK) (SB 203580). 4. Consistent with these results, MAFP caused membrane translocation of PKCbetaI and betaII isoforms and activated extracellular signal-regulated kinase (ERK) and p38 MAPK. 5. In accordance with these effects of MAFP, PKC activator phorbol 12-myristate 13-acetate (PMA) increased PGE2 release and caused activation of PKCbeta, ERKs and p38 MAPK. 6. This is the first report that the PLA2 inhibitor, MAFP, can induce COX-2 gene expression and PGE2 synthesis via the PKC-, ERK- and p38 MAPK-dependent pathways. Thus, the use of MAFP as a PLA2 inhibitor should be treated with caution.